A method for constructing a fingerprint spectrum of Shenkun Yangxue granules and the fingerprint spectrum thereof

Abstract

The application discloses a kind of Shenkun Yangxue granules fingerprint and its construction method, belong to traditional Chinese medicine detection technical field.Different batches of Shenkun Yangxue granules are prepared into test sample solution;Respectively with Danshensu, ephedrine hydrochloride, syringin, protocatechuic aldehyde, coffee acid, calycosin-7-O-β-D-glucoside, loganin, comphoric acid, shisonin, delphinidin and luteolin and kaempferol control preparation single control solution;Test sample solution and control solution are analyzed by high performance liquid chromatography and high resolution mass spectrometry respectively, 18 characteristic peaks are screened out, and 12 kinds of chemical components are identified by standard comparison and fragment information analysis, to obtain Shenkun Yangxue granules fingerprint.The Shenkun Yangxue granules fingerprint established by the method can be used for the quality control detection of Shenkun Yangxue granules, which is conducive to the comprehensive monitoring of product quality, and ensures the consistency, controllability and stability of product quality.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a construction method of a Shenkun Yangxue granule fingerprint and the fingerprint. BACKGROUND

[0002] Shenkun Yangxue granule is composed of six medicinal materials, i.e., Huangqi, Danshen, Dangshen, Danggui, Yimucao and Beipaijiang, and has the effects of benefiting qi and nourishing blood and promoting blood circulation to remove blood stasis, and is commonly used in clinic for treating postpartum lochia not dried up and symptoms of abdominal pain caused by qi deficiency and blood stasis.

[0003] In recent years, Shenkun Yangxue granule has been widely concerned as a traditional Chinese medicine preparation due to its significant curative effect and low side effects. However, due to the complex composition of traditional Chinese medicine and the multiple interaction mechanisms between components, the quality control of Shenkun Yangxue granule is a great challenge. At present, the quality research of Shenkun Yangxue granule is limited to the analysis of single medicinal component in the formula, and the overall composition is ignored. The current quality standard only includes the identification of medicinal materials in the formula by thin layer chromatography and the content determination of protocatechuic aldehyde by high performance liquid chromatography. However, the chemical composition of Shenkun Yangxue granule is complex, and only relying on the identification or content determination of a single compound cannot fully reflect the material basis and chemical composition information, and thus cannot realize the comprehensive control of its internal quality, which may affect the stability and safety of its clinical effect. Therefore, in order to improve the quality control level of Shenkun Yangxue granule, it is urgent to develop a more comprehensive and systematic detection technology to realize the comprehensive evaluation of the overall composition of the granule. SUMMARY

[0004] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a construction method of a Shenkun Yangxue granule fingerprint and the fingerprint, so as to solve the technical problem that the existing Shenkun Yangxue granule detection method cannot comprehensively control the quality.

[0005] In order to achieve the above purpose, the following technical solutions are adopted in the present application:

[0006] In a first aspect, the present application discloses a construction method of a Shenkun Yangxue granule fingerprint, comprising the following steps:

[0007] S1, preparing a test solution: different batches of Shenkun Yangxue granule fine powder are weighed, an extraction solvent is added, and reflux extraction is performed to obtain a test solution;

[0008] S2, preparing a control solution: Danshensu, Yimucao hydrochloride, syringin, protocatechuic aldehyde, caffeic acid, calycosin-7-O-β-D-glucoside, loganin, calycosin, rosmarinic acid, shikonin, luteolin and kaempferol are precisely weighed, dissolved, and a single control solution is obtained; S3, preparing a fingerprint sample: the test solution and the control solution are mixed to obtain a fingerprint sample; S4, constructing a fingerprint: the fingerprint sample is analyzed by high performance liquid chromatography-mass spectrometry to obtain a Shenkun Yangxue granule fingerprint.

[0009] S3, chromatographic analysis was performed on the test sample solution obtained in S1 and the control sample solution in S2 respectively, and the corresponding chromatograms were recorded;

[0010] S4, the chromatogram obtained in S3 was introduced into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system for similarity analysis;

[0011] S5, high resolution mass spectrometry was performed on the test sample solution obtained in S1 to obtain a total ion flow chart and a mass spectrum result chart of chemical components, the detection data was introduced into Xcalibur software, entered the Qual Browser interface, data analysis was performed according to the peak situation of chemical components, and the chemical components of each peak in the test sample chromatogram were determined according to the total ion flow chart and the mass spectrum result chart of chemical components combined with the control sample chromatogram, to obtain the Shenkun Yangxue Granules fingerprint.

[0012] Preferably, in S1, the extraction solvent is pure methanol solution.

[0013] Preferably, in S1, the reflux extraction is performed for 30 min.

[0014] Preferably, in S1, the ratio of the use amount of Shenkun Yangxue Granules fine powder to the extraction solvent is 3.5 g:25 mL.

[0015] Preferably, in S2, the preparation method of the control sample solution is: respectively precisely weighing Danshensu, marrubiin hydrochloride, syringin, protocatechuic aldehyde, coffee acid, calycosin-7-O-β-D-glucoside, loganin, mutamycin, rosmarinic acid, shikonin, luteolin and kaempferol control samples, adding pure methanol solution, and preparing a single control sample solution containing Danshensu 56 μg, marrubiin hydrochloride 27 μg, syringin 19 μg, protocatechuic aldehyde 37 μg, coffee acid 26 μg, calycosin-7-O-β-D-glucoside 11 μg, loganin 16 μg, mutamycin 89 μg, rosmarinic acid 25 μ, shikonin 49 μg, luteolin 6 μg or kaempferol 19 μg per 1 mL.

[0016] Preferably, in S3, the liquid chromatography conditions are: Hedera ODS-2-C18, 4.6×250 mm×5µm; DAD detector is used, the detection wavelength is 210 nm; the column temperature is 30℃; the injection amount is 10 μL;

[0017] Further preferably, in the liquid chromatography condition: the mobile phase is acetonitrile-0.01% phosphoric acid aqueous solution, the flow rate is 1.0 mL / min, and the gradient elution program is as follows: 0~5 min, acetonitrile volume 2%; 5~10 min, acetonitrile volume 2%~13%; 10~20 min, acetonitrile volume 13%~22%; 20~25 min, acetonitrile volume 22%~32%; 25~33 min, acetonitrile volume 32%~49%; 33~39 min, acetonitrile volume 49%~46%; 39~40 min, acetonitrile volume 46%~60%.

[0018] Preferably, in S5, the high-resolution mass spectrometry detection condition is: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300 DEG C, auxiliary gas temperature 300 DEG C, and the scan mode is full scan mode, and the mass-to-charge ratio scan range m / z is 100-1500.

[0019] Preferably, in S5, according to the total ion chromatogram and the mass spectrum result diagram of the chemical components, and in combination with the chromatogram of the reference substance, the chemical components of each peak in the chromatogram of the test sample are determined, and are as follows: the peak No. 3 is danshensu, the peak No. 4 is motherwort alkaloid hydrochloride, the peak No. 5 is syringin, the peak No. 6 is protocatechuic aldehyde, the peak No. 7 is caffeic acid, the peak No. 9 is calycosin-7-O-β-D-glucoside, the peak No. 11 is party glycoside, the peak No. 13 is mutanghuangsu, the peak No. 14 is rosemary acid, the peak No. 16 is borax acid, the peak No. 17 is luteolin, and the peak No. 18 is kaempferol, so that the fingerprint spectrum of the Shenkun Yangxue granules is obtained.

[0020] In a second aspect, the application discloses the fingerprint spectrum of the Shenkun Yangxue granules obtained by the construction method.

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] The application provides a construction method of a Shenkun Yangxue granule fingerprint spectrum, 1) when preparing the test sample solution, a reflux extraction method is selected, the extraction effect is good, peak information is more, and the composition of the chromatogram is relatively comprehensive; 2) when preparing the control sample solution, salvianic acid, marrubiin hydrochloride, syringin, protocatechuic aldehyde, coffee acid, calycosin-7-O-beta-D-glucoside, paeoniflorin, ononin, rosmarinic acid, shikonin, luteolin and kaempferol are used as the control samples, the Shenkun Yangxue granule fingerprint spectrum analysis and comparison can be provided with a benchmark, and the accuracy and reliability of the constructed spectrum are ensured; 3) the consistency and stability of the traditional Chinese medicine quality are ensured through similarity analysis; 4) the comprehensiveness of the method can be ensured by combining chromatographic analysis with high-resolution mass spectrometry analysis. The established HPLC fingerprint spectrum of the Shenkun Yangxue granule is obtained by simultaneously determining 15 batches of Shenkun Yangxue granules, 12 kinds of chemical components are identified by high-resolution mass spectrometry, and the obtained chromatogram has high similarity, so that the Shenkun Yangxue granule can be effectively and comprehensively detected. The stability, repeatability and precision are calculated according to the relative retention time and the relative peak area, and the results show that the RSD values of the relative retention time and the relative peak area are all less than 3%, indicating that the method has the characteristics of good stability, high precision and good repeatability, can accurately, clearly, comprehensively and objectively evaluate the quality of the Shenkun Yangxue granule, has significant importance and practical value for effectively controlling the quality of the Shenkun Yangxue granule and ensuring the clinical curative effect, and provides quality assurance for the clinical curative effect. The establishment of the fingerprint spectrum of the Shenkun Yangxue granule ensures the stable play of the curative effect, can further promote the safety and effectiveness of the Shenkun Yangxue granule, and provides strong protection for the clinical application and popularization of the Shenkun Yangxue granule.

[0023] ‌Further, when preparing the test sample solution, pure methanol solution is used as the extraction solvent, the chromatogram information of the extract is the most, the content of the components is the highest, and the extraction effect is the best.

[0024] Further, the reflux extraction is performed for 30 min, the extraction time can be shortened on the basis of ensuring the extraction effect, and the extraction cost is reduced.

[0025] Further, when performing chromatographic analysis, the wavelength is selected as 210 nm, the information amount contained in the chromatogram is the most comprehensive and the baseline is stable. The column temperature is selected as 30 DEG C, and the separation effect of each component is better.

[0026] Further, when performing chromatographic analysis, octadecylsilane-bonded silica gel is used as the filler, acetonitrile-0.01% phosphoric acid aqueous solution is used as the mobile phase, the number of peak out is more, the peak type and separation effect are better. When the flow rate is 1.0 ml / min, the separation degree of each peak of the fingerprint spectrum is better. When the gradient elution program is selected, the separation degree is good, the peak type is better, the baseline is stable, and the chromatographic information is complete. By using the specific elution condition, the separation effect of various effective components is obviously improved while the detection time is shortened, so that more characteristic peaks are contained in the fingerprint spectrum, and the spectrum information is greatly enriched. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the specific embodiments or prior art description will be briefly introduced as follows.

[0028] Figure 1 The chromatogram obtained in the preparation process of the Shengkun Yangxue Granules test sample solution according to the present application in Example 1;

[0029] Figure 2 The chromatogram of the Danshensu reference substance (A) and the mass spectrum of the Danshensu (B) according to the present application;

[0030] Figure 3 The chromatogram of the Leonurine Hydrochloride reference substance (A) and the mass spectrum of the Leonurine Hydrochloride (B) according to the present application;

[0031] Figure 4 The chromatogram of the Syringin reference substance (A) and the mass spectrum of the Syringin (B) according to the present application;

[0032] Figure 5 The chromatogram of the Protocatechuic Aldehyde reference substance (A) and the mass spectrum of the Protocatechuic Aldehyde (B) according to the present application;

[0033] Figure 6 The chromatogram of the Caffeic Acid (A) and the mass spectrum of the Caffeic Acid (B) according to the present application;

[0034] Figure 7 The chromatogram of the Calycosin-7-O-β-D-Glucoside reference substance (A) and the mass spectrum of the Calycosin-7-O-β-D-Glucoside (B) according to the present application;

[0035] Figure 8 The chromatogram of the Partyneoside reference substance (A) and the mass spectrum of the Partyneoside (B) according to the present application;

[0036] Figure 9 The chromatogram of the Mutamycin reference substance (A) and the mass spectrum of the Mutamycin (B) according to the present application;

[0037] Figure 10Reference substance chromatogram (A) and mass spectrum (B) of rosmarinic acid of the present application;

[0038] Figure 11 Reference substance chromatogram (A) and mass spectrum (B) of shikonin of the present application;

[0039] Figure 12 Reference substance chromatogram (A) and mass spectrum (B) of luteolin of the present application;

[0040] Figure 13 Reference substance chromatogram (A) and mass spectrum (B) of kaempferol of the present application;

[0041] Figure 14 Mass spectrum negative ion total ion current chromatogram of the Shenkun Yangxue Granules of the present application;

[0042] Figure 15 Mass spectrum positive ion total ion current chromatogram of the Shenkun Yangxue Granules of the present application;

[0043] Figure 16 Fingerprint chromatograms of 15 batches of the Shenkun Yangxue Granules of the present application;

[0044] Figure 17 Chromatogram obtained by optimizing the extraction method during preparation of the test sample solution of the present application;

[0045] Figure 18 Chromatogram obtained by optimizing the extraction time during preparation of the test sample solution of the present application;

[0046] Figure 19 Chromatogram obtained by optimizing the composition of the mobile phase in the chromatographic conditions of the present application;

[0047] Figure 20 Chromatogram obtained by optimizing the detection wavelength in the chromatographic conditions of the present application;

[0048] Figure 21 Chromatogram obtained by optimizing the flow rate in the chromatographic conditions of the present application;

[0049] Figure 22 Chromatogram obtained by optimizing the column temperature in the chromatographic conditions of the present application;

[0050] Figure 23 Chromatogram obtained by optimizing the elution program in the chromatographic conditions of the present application. DETAILED DESCRIPTION

[0051] For those skilled in the art to understand the characteristics and effects of the present application, the following only explains and defines the terms and phrases mentioned in the specification and claims. Unless otherwise specified, all technical and scientific words used herein have their usual meanings to those skilled in the art of the present application, and in case of conflict, the definition in the specification shall prevail.

[0052] The present application provides a method for constructing a fingerprint of Shengkun Yangxue granules, comprising the following steps:

[0053] S1, preparing a test solution: different batches of Shengkun Yangxue granules powder were weighed and added to an extraction solvent solution, and refluxed to obtain a test solution of Shengkun Yangxue granules;

[0054] The extraction solvent solution is pure methanol solution, and the dosage ratio of Shengkun Yangxue granules powder to pure methanol solution is 3.5 g:25 mL;

[0055] S2, preparing a control solution: each control was precisely weighed and added to a pure methanol solution to prepare a single control solution containing 56 μg of danshensu, 27 μg of motherwort alkaloid hydrochloride, 19 μg of syringin, 37 μg of protocatechuic aldehyde, 26 μg of caffeic acid, 11 μg of calycosin-7-O-β-D-glucoside, 16 μg of party care glycoside, 89 μg of mutamycin, 25 μg of rosemary acid, 49 μg of purple grass acid, 6 μg of luteolin, and 19 μg of kaempferol per 1 mL;

[0056] S3, the test solution obtained in S1 and the control solution in S2 were injected into a high performance liquid chromatograph for chromatographic analysis, and the liquid chromatography conditions were as follows: column type Hedera ODS-2-C18 (4.6×250 mm×5 µm); DAD detector was used, and the detection wavelength was 210 nm; column temperature was 30℃; flow rate was 1.0 mL / min; injection volume was 10 μL; mobile phase was acetonitrile (A)-0.01% phosphoric acid aqueous solution (B), and the gradient elution program was as follows: 0~5 min, 2% A; 5~10 min, 2%~13% A; 10~20 min, 13%~22% A; 20~25 min, 22%~32% A; 25~33 min, 32%~49% A; 33~39 min, 49%~46% A; 39~40 min, 46%~60% A, which can realize good separation degree of each chromatographic peak in the fingerprint, and record the corresponding chromatogram;

[0057] S4, import the chromatogram obtained in S3 into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, select the chromatographic peaks existing in the chromatograms of different batches of Shenkun Yangxue Granules as common peaks, and perform similarity analysis on the chromatogram of the test solution through data import, multi-point correction and data matching;

[0058] S5, the test solution was subjected to high-resolution mass spectrometry analysis, and the high-resolution mass spectrometry detection conditions were as follows: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300 ℃, auxiliary gas temperature 300 ℃, scanning mode full scan mode, mass-to-charge ratio scanning range m / z 100-1500, total ion current chromatogram and mass spectrum result chromatogram of chemical components were obtained. The detection data was imported into Xcalibur software, entered the Qual Browser interface, and data analysis was performed according to the peak conditions of chemical components, and the chemical components of each peak in the chromatogram of the test solution were determined according to the total ion current chromatogram and the mass spectrum result chromatogram of chemical components combined with the chromatogram of the reference substance, which were as follows: peak No. 3 was danshensu, peak No. 4 was motherwort alkaloid hydrochloride, peak No. 5 was syringin, peak No. 6 was protocatechuic aldehyde, peak No. 7 was coffee acid, peak No. 9 was calycosin-7-O-β-D-glucoside, peak No. 11 was party glycoside, peak No. 13 was mutamycin, peak No. 14 was rosemary acid, peak No. 16 was alkannin, peak No. 17 was luteolin, peak No. 18 was kaempferol, and the fingerprint of Shenkun Yangxue Granules was obtained.

[0059] The application will be further described in conjunction with specific examples. It should be understood that these examples are used to illustrate but not to limit the scope of the application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the application after reading the content of the application, and these equivalent forms also fall within the scope of the appended claims.

[0060] The following examples use the conventional equipment in the art. The experimental methods not specified in the following examples are usually carried out according to the conventional conditions, or according to the conditions recommended by the manufacturers. Various raw materials are used in the following examples, and unless otherwise specified, the conventional commercially available products are used, and the specifications are conventional specifications in the art.

[0061] 1. Instruments

[0062] The instruments used in the application are shown in Table 1.

[0063] Table 1 Instruments used in the application

[0064]

[0065] 2. Medicines and reagents

[0066] The 15 batches of Shen Kunyangxue granules used in the application were provided by Shaanxi Bailv Pharmaceutical Co., Ltd. (Table 2), and the prescription originated from the State Administration of Pharmaceutical Administration. The Surgical and Gynecological Booklet of National Chinese Medicine Standard Compilation [WS-11309 (ZD-1309)-2002-2012Z]; the control: rosmarinic acid control (batch number: C14900895, purity: 97%) was purchased from Shanghai Maikelin Biochemical Technology Co., Ltd.; the control of tanshinone (batch number: YJ0157, purity: 98%) was purchased from Jiangsu Yongjian Pharmaceutical Technology Co., Ltd.; the control of protocatechuic aldehyde (batch number: 20082402, purity: 99.75%) and the control of bartsin 7-O-β-D-glucoside (batch number: 21022604, purity: 98%) were purchased from Chengdu Pufide Biological Technology Co., Ltd.; the control of caffeic acid (batch number: PS010522, purity: 98%), the control of syringin (batch number: PS010261, purity: 98%), the control of onocrotaloside (batch number: PS000671, purity: 98%), the control of shikonin (batch number: PS001156, purity: 98%), and the control of kaempferol (batch number: PS011676, purity: 98%) were all purchased from Chengdu Pus Biological Technology Co., Ltd.; the reagents used in the application are shown in Table 3.

[0067] Table 2 sample used

[0068]

[0069] Table 3 reagent used

[0070]

[0071] The following is an example of Shen Kunyangxue granules, and the application will be described in detail through specific examples.

[0072] Example 1

[0073] A method for constructing a Shen Kunyangxue granule fingerprint spectrum, comprising the following steps:

[0074] S1, preparation of test solution: accurately weigh 3.5 g of 15 batches of Shen Kunyangxue granules, respectively, into a conical flask with a plug, add 25 mL of pure methanol solution, tightly plug, reflux extraction for 30 min, shake well, filter, take the filtrate through a 0.45 µm microporous filter membrane, and obtain the test solution of Shen Kunyangxue granules.

[0075] S2, Preparation of the control solution: each control substance (Danshensu, Leonuride Hydrochloride, Syringin, Protocatechuic Aldehyde, Caffeic Acid, Calycosin-7-O-β-D-glucoside, Paeonol, Mutamycin, Rosmarinic Acid, Shikonin, Luteolin and Kaempferol) was precisely weighed and added into pure methanol solution to prepare a single control solution containing Danshensu 56 μg, Leonuride Hydrochloride 27 μg, Syringin 19 μg, Protocatechuic Aldehyde 37 μg, Caffeic Acid 26 μg, Calycosin-7-O-β-D-glucoside 11 μg, Paeonol 16 μg, Mutamycin 89 μg, Rosmarinic Acid 25 μg, Shikonin 49 μg, Luteolin 6 μg and Kaempferol 19 μg per 1 mL.

[0076] S3, 10 μL of the test solution obtained in S1 and the control solution in S2 were precisely taken respectively and injected into the high performance liquid chromatograph for chromatographic analysis, and the corresponding chromatograms were recorded, as shown in Figure 1 and Figures 2-13 of FIG. (A).

[0077] The liquid chromatography conditions were as follows: chromatographic column: Hedera ODS-2-C18 (4.6 x 250 mm x 5 μm) chromatographic column; detector: DAD detector; detection wavelength: 210 nm; flow rate: 1.0 mL / min; injection volume: 10 μL; column temperature: 30°C; mobile phase: acetonitrile-0.01% phosphoric acid aqueous solution, gradient elution, elution program as shown in Table 11:

[0078] S4, the chromatogram obtained in S3 was imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System, and the chromatographic peaks present in the chromatograms of 15 batches of Shenkun Yangxue Granules were selected as common peaks. The chromatogram of the test solution was subjected to data import, multi-point correction and data matching, and similarity analysis was performed (Table 4), and the reliability of the confirmation result was confirmed.

[0079] Table 4 Similarity between each batch of Shenkun Yangxue Granules and the common mode

[0080]

[0081] S5, in order to determine the chemical components in the fingerprint, the above test solution was subjected to mass spectrometry analysis. The high-resolution mass spectrometry detection conditions were as follows: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300°C, auxiliary gas temperature 300°C, scanning mode full scan mode, mass-to-charge ratio scanning range m / z 100-1500, to obtain the total ion current chromatogram and the mass spectrometry result chromatogram of the chemical components (FIG. (B), Figures 2-13 FIG. (C), Figure 14 and Figure 15 ).

[0082] S6. Import the detection data into Xcalibur software, enter the Qual Browser interface, and analyze the data based on the peaks of the chemical components. Determine the chemical components of each peak in the chromatogram of the test sample based on the total ion chromatogram, the mass spectrometry results of the chemical components, and the chromatogram of the reference standard. Peak 3 is tanshinone, retention time 14.357 min; peak 4 is leonurine hydrochloride, retention time 15.167 min; peak 5 is syringin, retention time 16.793 min; peak 6 is protocatechuic aldehyde, retention time 17.950 min; peak 7 is caffeic acid, retention time 20.360 min; peak 9 is 7-O-β-D-glucoside of verbascoside, retention time 24.140 min; peak 11 is codonopsis glycoside, retention time 28.137 min; peak 13 is gentianin, retention time 29.347 min; peak 14 is rosmarinic acid, retention time 29.677 min. Peak 16 is shikonin, retention time 31.823 min; peak 17 is luteolin, retention time 32.227 min; peak 28 is kaempferol, retention time 35.370 min, thus obtaining the fingerprint spectrum of Shenkun Yangxue Granules ( Figure 16 ).

[0083] S7. Methodological Investigation

[0084] 1. Precision Experiment

[0085] The test solution obtained in S1 was injected in parallel 6 times under the chromatographic conditions in S3, with an injection volume of 10 μL. Tanshinone, leonurine hydrochloride, syringin, protocatechuic aldehyde, caffeic acid, verbascoflavonoid 7-O-β-D-glucoside, codonopsis glycoside, gentianin, rosmarinic acid, shikonin, luteolin and kaempferol were used as reference peaks. The relative retention time and relative peak area were analyzed, and the RSD value was calculated. The results showed that the RSDs were 0.18%~0.33% and 0.84%~2.42%, respectively. The RSDs were all less than 3%, indicating that the instrument had good precision.

[0086] 2. Stability test

[0087] Take the test sample solution obtained in S1, and analyze it by sampling at 0 h, 2 h, 4 h, 8 h, 12 h and 24 h according to the chromatographic conditions in S3, with 10 μL of sample injection, and with danshensu, leucocyanidol hydrochloride, syringin, protocatechuic aldehyde, coffee acid, calycosin-7-O-β-D-glucoside, loganin, mutamycin, rosmarinic acid, shikonin, luteolin and kaempferol as reference peaks. The relative retention time and relative peak area of the common peaks in the sample HPLC fingerprint are analyzed, and the RSD value is calculated. The results show that the RSD is 0.35%~0.69% and 0.93%~2.31%, respectively, and the RSD is less than 3%, indicating that the test sample solution of Shenkun Yangxue Granules has good stability within 24 h.

[0088] 3. Repetitive experiment

[0089] Take 6 test sample solutions of Shenkun Yangxue Granules, and analyze them by sampling according to the chromatographic conditions in S3, with danshensu, leucocyanidol hydrochloride, syringin, protocatechuic aldehyde, coffee acid, calycosin-7-O-β-D-glucoside, loganin, mutamycin, rosmarinic acid, shikonin, luteolin and kaempferol as reference peaks. The relative retention time and relative peak area of the common peaks in the sample HPLC fingerprint are analyzed, and the RSD value is calculated. The results show that the RSD is 0.28%~0.71% and 0.66%~2.79%, respectively, and the RSD is less than 3%, indicating that the method has good repeatability.

[0090] Example 2

[0091] Optimization of test sample solution preparation: The purpose of this example is to investigate the influence of different extraction methods (ultrasonic, reflux, immersion) on the detection of test sample solution of Shenkun Yangxue Granules. The chromatographic conditions are the same as in Example 1, and the preparation conditions of the rest of the test sample solution are the same as in Example 1. The detection results are shown in Figure 17 It can be seen from Figure 17 that the extraction method is reflux, and the extraction effect is better, with more peak information and more comprehensive chromatogram composition.

[0092] Example 3

[0093] Optimization of test sample solution preparation: The purpose of this example is to investigate the influence of different extraction times (30 min, 60 min, 90 min) on the detection of test sample solution of Shenkun Yangxue Granules. The chromatographic conditions are the same as in Example 1, and the preparation conditions of the rest of the test sample solution are the same as in Example 1. The detection results are shown in Figure 18 It can be seen from Figure 18 that the detection results of extraction times of 30 min, 60 min and 90 min have little difference. Given that 30 min is more time-saving and cost-saving, the extraction time of 30 min is selected as the extraction time condition.

[0094] Example 4

[0095] Optimization of the preparation of the test solution: The purpose of this example is to investigate the effect of different extraction solvents (methanol, ethanol, acetonitrile, water, 0.01% phosphoric acid water) on the detection of the test solution of Shenkun Yangxue Granules. The chromatographic conditions are the same as in Example 1, and the preparation conditions of the remaining test solution are the same as in Example 1. The detection results show that the extraction effect of methanol is the best.

[0096] Example 5

[0097] Optimization of the preparation of the test solution: The purpose of this example is to investigate the effect of different extraction solvent concentrations (50% methanol solution, 80% methanol solution, pure methanol solution) on the detection of the test solution of Shenkun Yangxue Granules. The chromatographic conditions are the same as in Example 1, and the preparation conditions of the remaining test solution are the same as in Example 1. The detection results show that when pure methanol solution is used as the extraction solvent, the chromatogram of the extract contains the most information and has the highest component content.

[0098] Example 6

[0099] Optimization of the chromatographic conditions: The purpose of this example is to investigate the effect of different mobile phases (methanol-0.01% phosphoric acid, acetonitrile-0.1% phosphoric acid, acetonitrile-0.05% phosphoric acid, acetonitrile-0.02% phosphoric acid, acetonitrile-0.01% phosphoric acid, acetonitrile-0.05% acetic acid, acetonitrile-0.1% formic acid) on the detection of the test solution of Shenkun Yangxue Granules. The test solution is prepared by the method of Example 1, and the detection results are shown in Table 1. Figure 2 As can be seen from Table 1, when the mobile phase is acetonitrile-0.01% phosphoric acid, the number of peaks is larger, and the peak shape and separation effect are better. Figure 19

[0100] Example 7

[0101] The purpose of this example is to investigate the effect of different wavelengths (190 nm, 210 nm, 245 nm, 275 nm, 280 nm, 310 nm) on the detection of the test solution of Shenkun Yangxue Granules. The test solution is prepared by the method of Example 1, and the remaining chromatographic conditions are the same as in Example 1. The detection results are shown in Table 2. Figure 20 As can be seen from Table 2, when the wavelength is 210 nm, the chromatogram contains the most comprehensive information and the baseline is stable, so this method is selected as the detection wavelength condition. Figure 20

[0102] Example 8

[0103] ​​The purpose of this embodiment is to investigate the effect of different flow rates (0.6 ml / min, 0.8 ml / min, 1.0 ml / min) on the detection of the test solution. The test solution was prepared using the method of Example 1, and all other chromatographic conditions were the same as in Example 1. The detection results are as follows: Figure 21 As shown. (Through) Figure 21 It can be seen that the separation of each peak in the fingerprint spectrum is better when the flow rate is 1.0 ml / min, so the flow rate of 1.0 ml / min is selected as the flow rate condition.

[0104] Example 9

[0105] The purpose of this embodiment is to investigate the effect of different column temperatures (25℃, 30℃, 35℃) on the detection of the Shenkun Yangxue Granules test solution. The test solution was prepared using the method in Example 1, and all other chromatographic conditions were the same as in Example 1. The detection results are as follows: Figure 22 As shown. (Through) Figure 22 It can be seen that the separation effect of each component is better when the column temperature is 30℃, so the column temperature of 30℃ is selected.

[0106] Example 10

[0107] The purpose of this embodiment is to investigate the effect of different elution programs on the detection of the Shenkun Yangxue Granules test solution. The test solution was prepared using the method in Example 1, and elution programs were set for each solution. All other chromatographic conditions were the same as in Example 1. Some elution programs are shown in Tables 5 to 11.

[0108] Table 5 Elution Procedure 1

[0109]

[0110] Table 6 Elution Procedure 2

[0111]

[0112] Table 7 Elution Procedure 3

[0113]

[0114] Table 8 Washing Procedure 4

[0115]

[0116] Table 9 Washing Procedure 5

[0117]

[0118] Table 10 Elution Procedure 6

[0119]

[0120] Table 11 Elution Procedure 7

[0121]

[0122] Test results as follows Figure 23 As shown. (Through) Figure 23 It can be seen that elution program 7 has good separation, better peak shape, stable baseline and complete chromatographic information. Therefore, elution program 7 is selected as the optimal elution program.

[0123] The above experimental results show that this method has the characteristics of good stability, high precision and good repeatability, and can comprehensively and objectively evaluate the quality of Shenkun Yangxue Granules, providing quality assurance for clinical efficacy.

[0124] This invention, through screening extraction solvents, extraction methods, mobile phases, column temperatures, and flow rates, uses octadecylsilane-bonded silica gel as the packing material and acetonitrile and phosphoric acid aqueous solution as the mobile phase. Employing specific elution conditions, it significantly improves the separation of multiple active ingredients while shortening the detection time. This results in a fingerprint spectrum containing more characteristic peaks, greatly enriching the spectral information. Furthermore, high-resolution mass spectrometry identifies 12 chemical components, enabling effective and comprehensive detection of Shenkun Yangxue Granules. This invention establishes for the first time a fingerprint spectrum quality evaluation method for Shenkun Yangxue Granules. Using this method, the types and quantities of the main chemical components contained in the traditional Chinese medicine Shenkun Yangxue Granules can be comprehensively detected, providing a rapid, convenient, and accurate objective and comprehensive assessment of the quality of Shenkun Yangxue Granules.

[0125] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for constructing a fingerprint spectrum of Shenkun Yangxue Granules, characterized in that, Includes the following steps: S1. Preparation of test solution: Weigh different batches of Shenkun Yangxue Granules fine powder, add methanol and reflux to extract, and obtain test solution; S2. Preparation of reference solution: Accurately weigh tanshinone, leonurine hydrochloride, syringin, protocatechuic aldehyde, caffeic acid, verbascoflavonoid 7-O-β-D-glucoside, codonopsis glycoside, gentianin, rosmarinic acid, lithospermic acid, luteolin and kaempferol, dissolve them to obtain a single reference solution. S3. The test solution obtained in S1 and the reference solution obtained in S2 were subjected to chromatographic analysis. The chromatographic conditions were as follows: Hedera ODS-2-C18 column, 4.6×250 mm×5 µm; DAD detector, detection wavelength 210 nm; column temperature 30℃; injection volume: 10 μL; mobile phase: acetonitrile-0.01% phosphoric acid aqueous solution; flow rate: 1.0 mL / min; gradient elution program: 0~5 min, acetonitrile volume 2%; 5~10 min, acetonitrile volume 2%~13%; 10~20 min, acetonitrile volume 13%~22%; 20~25 min, acetonitrile volume 22%~32%; 25~33 min, acetonitrile volume 32%~49%; 33~39 min, acetonitrile volume 49%~46%; 39~40 min, acetonitrile volume 46%~60%. The corresponding chromatograms were recorded. S4. Import the chromatogram obtained in S3 into the Chinese herbal chromatographic fingerprint similarity evaluation system for similarity analysis; S5. Perform high-resolution mass spectrometry analysis on the test solution obtained in S1 to obtain the total ion chromatogram and the mass spectrometry results of the chemical components. Import the detection data into Xcalibur software, enter the Qual Browser interface, and perform data analysis based on the peaks of the chemical components. Based on the total ion chromatogram and the mass spectrometry results of the chemical components, and in combination with the chromatogram of the reference standard, determine the chemical components of each peak in the chromatogram of the test sample to obtain the fingerprint spectrum of Shenkun Yangxue Granules.

2. The method for constructing the fingerprint spectrum of Shenkun Yangxue Granules according to claim 1, characterized in that, In S1, reflux extraction was performed for 30 min.

3. The method for constructing the fingerprint spectrum of Shenkun Yangxue Granules according to claim 1, characterized in that, In S1, the ratio of fine powder of Shenkun Yangxue Granules to extraction solvent is 3.5 g: 25 mL.

4. The method for constructing the fingerprint spectrum of Shenkun Yangxue Granules according to claim 1, characterized in that, In S2, the preparation method of the reference solution is as follows: accurately weigh tanshinone, leonurine hydrochloride, syringin, protocatechuic aldehyde, caffeic acid, verrucoside 7-O-β-D-glucoside, codonopsis glycoside, gentianin, rosmarinic acid, shikonin, luteolin, and kaempferol reference standards respectively, add them to pure methanol solution to prepare a single reference solution containing 56 μg tanshinone, 27 μg leonurine hydrochloride, 19 μg syringin, 37 μg protocatechuic aldehyde, 26 μg caffeic acid, 11 μg verrucoside 7-O-β-D-glucoside, 16 μg codonopsis glycoside, 89 μg gentianin, 25 μg rosmarinic acid, 49 μg shikonin, 6 μg luteolin or 19 μg kaempferol per mL.

5. The method for constructing the fingerprint spectrum of Shenkun Yangxue Granules according to claim 1, characterized in that, In S5, the high-resolution mass spectrometry detection conditions are: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300℃, auxiliary gas temperature 300℃, scanning mode is full scan mode, and mass-to-charge ratio scanning range m / z is 100-1500.

6. The method for constructing a fingerprint spectrum of Shenkun Yangxue Granules according to claim 1, characterized in that, In S5, based on the total ion chromatogram and the mass spectrometry results of the chemical components, combined with the chromatogram of the reference standard, the chemical components of each peak in the chromatogram of the test sample were determined as follows: peak 3 is tanshinone, peak 4 is leonurine hydrochloride, peak 5 is syringin, peak 6 is protocatechuic aldehyde, peak 7 is caffeic acid, peak 9 is 7-O-β-D-glucoside of verbascoside, peak 11 is codonopsis glycoside, peak 13 is gentianin, peak 14 is rosmarinic acid, peak 16 is shikonin, peak 17 is luteolin, and peak 18 is kaempferol. The fingerprint spectrum of Shenkun Yangxue Granules was thus obtained.

7. The fingerprint spectrum of Shenkun Yangxue Granules obtained by the construction method according to any one of claims 1 to 6.

Images